ABSTRACT Human epidermis serves as a critical barrier against mechanical, chemical, radiation and infectious insults in the external environment. Keratinocytes are the main cell type in this stratified tissue, and are organized into basal stem and progenitor cells, and the more superficial differentiated progeny. Epidermal homeostasis and integrity are tightly regulated and critical for human health. Squamous cell carcinomas (SCCs) are perhaps a prime example of epidermal homeostasis gone awry. Both sporadic and inherited forms of SCC are associated with mutations in the 21 genes of the Fanconi anemia (FA) pathway, which participate in the repair of DNA crosslinks. Close associations between the FA pathway and SCC development point to important roles for this pathway in keratinocytes and epidermis ? roles that have not yet been studied, but will be investigated here using FA patient populations and tools to study keratinocyte biology. These tools include human iPSC-derived keratinocytes and 3D organotypic epithelial rafts isogenic for FA genes, as well as skin biopsies from FA patients (vs. controls) that are uniquely available at Cincinnati Children's Hospital. Our translational studies build on recent data demonstrating cell adhesion, cytoskeletal and membrane defects in FA-deficient keratinocytes, skin biopsies, and 3D organotypic rafts. The defective keratinocytes were also hyper-motile, and invasive particularly when transformed ? and the invasive phenotype depended on the activities of the DNA damage sensor kinase DNA-PK and the small GTPase Rac1, as well as ganglioside accumulation. The overarching hypothesis is that FA pathway loss leads to the assembly of a Rac1 signalosome in membrane domains enriched for gangliosides (eg lipid rafts), and consequent phenotypes that are reminiscent of epithelial-mesenchymal-transition (EMT-like). Aim 1 tests the hypothesis that the FA pathway regulates adhesion/integrity and cell motility in the normal epidermis through ganglioside accumulation and DNA- PK/Rac1 signaling. Aim 2 directly tests the impact of FA loss on transformation, defines the FA-pathway- dependent flux of ganglioside biosynthesis and its role in sustaining SCC growth and progression. The results will identify a) signaling and lipid-based mechanisms whereby nuclear DNA repair machineries control the integrity of human epidermis, and b) biomarkers and therapeutic targets for the prevention and treatment of SCC development in patients with acquired and inherited FA pathway deficiencies.